Dual targeted proteins represent a problem for simple genetic analysis by gene knockout or by shRNA knockdown, since under such treatments both isoproteins are depleted. The TCA enzyme fumarase (FH) is dual targeted to the cytosol and mitochondria in all eukaryotic cells. While the mitochondrial function of fumarase is well known, the cytosolic function is unclear. Being a scavenger of fumarate made in the urea cycle or from the degradation of certain amino acids could not really explain the evolutionary conserved large amounts of the enzyme in the cytosol. Creation of a yeast strain, in which fumarase is synthesized only within mitochondria, allowed us to screen for unique phenotypes due to the lack of fumarase in the cytosol. Here we report that the absence of fumarase from the cytosol makes the yeast strains more sensitive to DNA damage, mainly to DSBs. This is also true for human FH, as cells knocked down for FH are more sensitive to DSBs. Data supporting this finding include:
(1) Strains of yeast and human cell cultures knocked down for fumarase-FH are sensitive to DNA damage; (2) fumarase in yeast and FH in human cells are induced upon generation of DNA damage; (3) a single amino acid mutation, which abolishes the enzymatic activity of fumarase, renders yeast more sensitive to DSBs; (4) the cellular location of FH is shifted rapidly to the nucleus upon DNA damage induction; (5) cytosolic absence of fumarase in yeast partially suppresses the DNA damage sensitive phenotype of the DNA repair genes, rad10 and rev3; and (6) in the absence of FH, phosphorylation of histone H2AX and of CHK2 and cell cycle checkpoint activation after DSBs induction are impaired.
It was not the initial objective of this study to determine the precise role of fumarase-FH in the DNA damage response. Yet our results may provide some clues to the nature of its extra-mitochondrial activity. Fumarase-FH, according to the phenotypes of its absence, appears to be involved in the cellular response to DNA DSBs. This is supported by the fact that upon induction of DNA damage, FH re-localizes to the nucleus. Notably, it does not accumulate in discrete nuclear foci, at sites of the breaks, as many members of the DNA damage response ( and ) 
. Moreover, in the absence of FH, phosphorylation of H2AX and CHK2 and cell cycle checkpoint activation are impaired, suggesting that FH may play a role in the cellular response to DSBs. Important evidence for our current model were the findings that the enzymatic activity of fumarase is essential for its protection from DSBs and that externally added high concentrations of fumaric acid can complement the absence of fumarase in the nucleus/cytosol and protect cells from DSBs. Nevertheless, following IR the whole cell levels of fumaric acid did not change significantly (Figure S3
). Taken together these results suggest that FH is not a DNA repair enzyme/protein but rather plays a role in the detection or signaling of DNA damage and in the maintenance of the DSB response machinery. We suggest that FH is doing so by determining the local level of fumaric acid in the nucleus. Unfortunately there is no known way to determine metabolite levels of small molecules such as fumarate or malate in the nucleus. The fact that malic acid cannot and fumaric acid can protect the cells from DSBs brings us to suggest that the conversion of malic to fumaric acid is the relevant function of fumarase in the nucleus. Our finding that there are no significant changes in the whole cell levels of these metabolites following DNA damage suggests that probably the levels of fumaric acid, formed locally by the action of fumarase, in sub-locations of the nucleus are important.
We propose the following model: under normal conditions FH is dual localized in the cytosol and mitochondria where, in the latter, it participates in the TCA cycle. The cytosolic population of FH molecules constitutes a pool, after which the induction of DNA damage (DSBs) is recruited to the nucleus. In the nucleus FH locally produces fumaric acid (from malic acid), which plays a role in the sensing, regulation, and/or stability of the DNA damage response machinery.
The findings indicating that FH, a mitochondrial metabolic enzyme, is a tumor suppressor were fascinating. In this regard, implication of FH in the DNA damage response, in this study, can in essence explain this tumor suppressor activity. In other words, genes involved in the DNA damage response are prime candidates to be tumor suppressors, because in the absence of an intact response (e.g., impaired repair or checkpoint activation) tumorogenic mutations can be established 
. A recent important report has suggested that in some cases of FH deficiency, there is no accumulation fumarate nor stabilization and accumulation of HIF 
. This observation can now be explained by a second cellular function of FH in the DNA damage response. We suggest that the major function of FH as a tumor suppressor gene is due to its role in the cellular response to DNA DSBs.
This study shows for the first time an exciting connection between primary metabolism (represented by the enzyme fumarase and its corresponding metabolite, fumaric acid) and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation. Our findings also support the notion that metabolic enzymes, in addition to being crucial agents of anabolism and catabolism, may play additional, oftentimes central, roles in other cellular activities, as for example aconitase in nucleoid binding and stabilization of mitochondrial DNA 
, aconitase as a regulator of iron metabolism 
, and more recently pyruvate kinase M2 as a phospho-tyrosine binding protein that is critical for a change in metabolism and rapid growth of cancer cells